The present invention is directed to a method and apparatus for performing isoelectric focusing of macromolecules, and particularly proteins. More particularly, the present invention is directed to an automated apparatus for the first dimension isoelectric focusing of proteins.
Isoelectric focusing (IEF) is an electrophoretic technique for the analysis, separation and purification of various biological materials. Since many of the complex molecules of biological interest are amphoteric in nature, they are typically amenable to IEF separation.
Isoelectric separation is a known process that has been used for many years. An isoelectric focusing gel, such as acrylamide, is placed or polymerized in a tube and positioned in a bath with a buffer solution at each end. One buffer solution is typically a sodium hydroxide solution. The other buffer solution is typically a phosphoric acid solution. When current is applied, the two buffer solutions, together with ampholytes incorporated into the gel composition or titratable gel monomers incorporated into the gel, provide a pH gradient through the gel along the length of the tube. The sample to be analyzed is applied to a top end of the gel in a tube and an electric current is applied to an electrode in each of the buffer solutions. The molecules in the sample migrate through the gel under the influence of the electric potential until they reach their isoelectric point.
The separation of macromolecules, and particularly proteins, often is carried out by two-dimensional electrophoresis separations. The two-dimensional electrophoresis separation typically involves the sequential separation by isoelectric focusing of a sample in a gel tube followed by slab gel electrophoresis. The isoelectric focusing process is often referred to as first dimension separation. Slab gel electrophoresis, often referred to as second dimension separation, utilizes an electrophoresis gel molded between two glass plates. A gel strip or cylinder in which the protein sample has been resolved by the first dimension isoelectric focusing is placed along one edge of the slab gel. The proteins are then allowed to migrate through the gel slab under an applied voltage.
Charged detergents, such as sodium dodecyl sulfate, contained in the slab gel bind to the protein molecules. The detergents tend to unfold the protein molecules into rods having a length proportional to the length of the polypeptide chain and thus proportional to the molecular weight of the polypeptide. A protein complexed with a charged detergent is highly charged, which causes the protein-detergent complex to move in an applied electric field. When the slab gel, such as a polyacrylamide gel, functions as a sieve, the movement of the longer and higher molecular weight molecules is retarded compared to the shorter, lower molecular weight molecules.
Electrophoresis separation is generally labor intensive since numerous samples are run simultaneously. Generally, the gel tubes are prepared and placed in a suitable tank of buffer solutions. The protein samples are then manually placed on the end of a gel tube. When hundreds of protein samples are prepared daily for isoelectric focusing, the manual steps significantly increase the time requirements for performing the first dimension separation. Accordingly, there is a need in the industry for improved methods and devices for conducting first dimensional isoelectric focusing.
The present invention is directed to an apparatus for the electrophoresis separation of macromolecules and particularly proteins. More particularly, the invention is directed to an apparatus for first dimensional isoelectric focusing of proteins and other macromolecules.
Accordingly, a primary object of the invention is to provide an automated apparatus for preparing samples for electrophoresis separation.
Another object of the invention is to provide an automated apparatus for sequentially transferring a biological sample from a sample container to a gel tube for performing electrophoresis separation of the sample.
A further object of the invention is to provide an automated apparatus for transferring a biological sample from a sample container to a gel tube where the sample container is identified and selected from a container supply magazine.
Another object of the invention is to provide an automated apparatus for electrophoresis separation including a sample container magazine having a holding device for holding a sample container while a sample is being removed.
A further object of the invention is to provide an automated apparatus for electrophoresis separation including a computer controlled arm having a pipette for piercing a septum in a sample container and removing a sample from the container.
Still another object of the invention is to provide an automated apparatus for electrophoresis separation including a computer controlled arm having a pipette, and a sample container holding device for holding the container while the pipette penetrates and is withdrawn from a septum in a sample container.
Another object of the invention is to provide an automated apparatus for transferring a plurality of sample solutions to a respective gel tube and recording and tracking the location of the samples.
A further object of the invention is to provide an automated apparatus for transferring a plurality of sample solutions to a respective gel tube, wherein the apparatus includes a pipette mounted on an arm that is movable vertically for withdrawing a sample from a container and for dispensing a sample to a gel tube.
Another object of the invention is to provide an automated apparatus for electrophoresis separation having a movable robotic arm and a pipette that is movable from a sample withdrawing position to a sample dispensing position.
A further object of the invention is to provide an automated apparatus for electrophoresis separation having a movable robotic arm, where movement of the arm actuates a holding device for holding a sample container while a sample is withdrawn from the sample container.
Another object of the invention is to provide a rack for supporting a plurality of gel tubes for electrophoresis separation, wherein the rack includes a guide for guiding a pipette to a gel tube.
Still another object of the invention is to provide a rack for supporting a plurality of gel tubes, where the rack includes a top and bottom wall defining a chamber, a top wall having a plurality of inlets having a guide surface, and the bottom wall having a plurality of openings with a guide surface aligned with a respective inlet in the top wall for guiding a pipette through said chamber to a respective gel tube.
A further object of the invention is to provide a rack for supporting a plurality of gel tubes for electrophoresis separation including a pair of electric contacts received in a pair of complementary recesses in a gel tank for positioning the rack in a predetermined location in said tank.
Another object of the invention is to provide an electrophoresis separation apparatus having a computer for controlling electric power supply to the gel tanks and for the acquisition of run data for quality control.
The foregoing objects and advantages of the invention are basically attained by providing an automated first dimensional electrophoresis separation apparatus comprising: an electrophoresis assembly including a tank, a rack positionable in the tank, a plurality of gel tubes containing an electrophoretic gel and being supported by the rack. Each of the tubes has a first open end and second open end. The rack includes a chamber for containing a first buffer solution and is in communication with the first end of the tubes. The tank is dimensioned for containing a second buffer solution in contact with the second end of the tubes. An electrical power source is connected to a first electrode in the chamber for contacting the first solution and a second electrode in the tank for contacting the second solution. The apparatus contains a supply magazine for containing a plurality of sample containers where each sample container contains a sample to be subjected to electrophoresis. The apparatus also contains a transferring device for sequentially removing a sample from a preselected sample container and transferring the sample to a first end of a respective gel tube, and a microprocessor for controlling the transferring device and automatically controlling the transfer of the samples to the respective gel tubes.
The objects of the invention are further attained by providing an automated electrophoresis apparatus comprising: an electrophoresis assembly including a tank, a rack removably positioned in the tank, a plurality of gel tubes containing an electrophoretic gel and being supported by the rack, each of the tubes having a first open end and second open end, the rack having a chamber for containing a first buffer solution in contact with the first end of the tubes, the tank being dimensioned for containing a second buffer solution in contact with the end of the tubes, and an electric power source having a first electrode in the chamber for contacting the first solution and a second electrode in the tank for contacting the second solution; a supply magazine for containing a plurality of sample containers, each sample containing a sample to be subjected to electrophoresis separation; a movable arm having a pipette, a vacuum source operatively connected to the pipette, and a pressure source operatively connected to the pipette, the arm being movable from a first position where the pipette removes a sample from a preselected sample container to a second position where the pipette dispenses the sample to a preselected gel tube; and a microprocessor connected to the arm for controlling movement of the arm and actuating the vacuum source when the pipette is in the first position, and to actuate the pressure source when the pipette is in the second position to sequentially transfer a sample from the sample containers to a respective gel tube.
The objects of the invention are still further attained by providing an electrophoresis assembly comprising: a tank for containing a buffer solution, the tank having at least one side wall with a top end having at least two spaced-apart apertures therein; a rack removably positioned in the tank, the rack having an upper end, a lower end, a chamber formed in the upper end for containing a second buffer solution, the chamber having a top wall and a bottom wall, the top wall and the bottom wall having a plurality of spaced-apart aligned openings, and at least two spaced-apart pins complementing the apertures in the side wall of the tank and for orienting the rack in a predetermined location in the tank; and a plurality of gel tubes, each of the tubes having a first end received in a respective opening in the bottom wall of the chamber.
The objects of the invention are yet further attained by providing a rack for supporting a plurality of gel tubes in an electrophoresis assembly, the rack comprising: at least one support member for supporting the rack in an electrophoresis tank; a top wall having a plurality of spaced-apart openings; a bottom wall having a plurality of spaced-apart openings aligned with a respective opening in the top wall; and at least one side wall, where the at least one side wall, top wall and bottom wall form a chamber for containing an electrophoresis buffer solution.
The objects of the invention are further obtained by providing a method for automated first dimensional electrophoresis separation comprising the steps of: providing a plurality of samples in sample containers; robotically removing the samples from the sample containers; robotically delivering the samples to one end of a respective isoelectric focusing gel; and controlling an electrical current to the isoelectric focusing gel and separating portions of the samples within the isoelectric focusing gel.
The objects of the invention are yet further obtained by providing an automated first dimensional electrophoresis separation apparatus comprising: an electrophoresis assembly including a tank, a rack positionable in the tank, a plurality of gel tubes containing an isoelectric focusing gel and being supported by the rack, the rack including at least two alignment pins, the alignment pins being received in alignment apertures formed in a portion of the tank for insuring precise positioning of the rack in the tank; a supply magazine for containing a plurality of sample containers, each sample container containing a sample to be subjected to electrophoresis; a transferring device for sequentially removing a sample from a pre-selected sample container and transferring the sample to a first end of a respective gel tube; and a microprocessor for controlling the transferring device and automatically controlling the transfer of the samples to the respective gel tubes.
The objects of the invention are still further obtained by providing an automated first dimensional electrophoresis separation apparatus comprising: an electrophoresis assembly including a tank, a rack positionable in the tank, a plurality of gel tubes containing an isoelectric focusing gel and being supported by the rack; a supply magazine for containing a plurality of sample containers, each sample container containing a sample to be subjected to electrophoresis separation; a transferring device for sequentially removing a sample from a preselected sample container and transferring the sample to a first end of a respective gel tube; a container holding member for holding a pre-selected sample container while the transferring device removes a sample from the sample container; and a microprocessor for controlling the transferring device and automatically controlling the transfer of the samples to the respective gel tubes.
The objects, advantages and salient features of the invention will become apparent to one skilled in the art in view of the following detailed description of the invention in conjunction with the annexed drawings which form a part of this original disclosure.